Bacillus anthracis: A recombinant PA from an uncapsulated strain and several formaldehyde-treated and/or alum-adsorbed formulations were prepared and injected 3 times, 2 months apart, followed by another injection 1 year later into adult volunteers. All formulations were safe and local and systemic reactions were rare and minor. Antibody assays compared favorably with those of the licensed vaccine. Peptides of the homopolymer of D-gamma-glutamic acid (D-G-PGA) were bound to rPA or TT. Chimpanzees were immunized sc with rPA-PGA or TT-PGA (10 mcg PGA/animal). Both chimps responded with antibodies to both vaccine components. Higher anti-PGA levels were obtained with the TT conjugate. Five D-G-PGA-specific Fabs were generated from immunized chimpanzees. Two were selected for further study and converted into full-length human constant region IgG1 and IgG3 monoclonal antibodies (mAb). A single 30 mcg dose of either mAb, given to BALB/c mice 18 h before intratracheal spore challenge with the virulent B. anthracis Ames strain, conferred protection from about 40 LD50. Also, both mAb given 8 h or 20 h after challenge provided significant protection. Thus, these anti-D-G-PGA mAbs would be useful, alone or in combination with anti-toxin mAbs, for a safe and efficacious postexposure therapy for anthrax. Plasmodium falciparum: The most studied experimental malaria vaccines have been the circumsporozoite protein (CSP), expressed on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were safe and mildly immunogenic, but their protection was poor and of limited duration even when administered with adjuvants. We used two approaches to provide experimental malaria vaccines: 1. The first is directed to the sexual, mosquito parasite stage, to provide a transmission blocking vaccine. Pfs25, a low molecular mass protein, non immunogenic by itself, was bound onto itself or to carrier proteins by amide, hydrazone or thioether linkages. Injected into mice, all conjugates were immunogenic with booster responses upon reinjection. Long term studies revealed a unique property of Pfs25 bound onto itself; IgG antibody levels increased with time, peaking at around 7 months and starting to decline at 9. Antibody levels of Pfs25 conjugated to other carriers started to decline after 3 months. The best immunogens used adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum increased the antibody levels further. Transmission blocking activity of immune sera correlated with antibody levels measured by ELISA. Similar results were obtained with Pvs25-Pvs25 (P. vivax) conjugates. 2. The second follows earlier studies using NANP (Asn-Ala-Asn-Pro), the repeat fragment of the circumsporozoite protein (CSP) of the pre-erythrocytic parasite stage as a vaccine. Four and 5 NANP repeats were synthetized and conjugated to BSA. These conjugates were immunogenic in mice, induced booster responses with corresponding high titers in Immuno Fluorescent Assay (IFA). The addition of a CSP T-cell epitope to the NANP repeats did not enhance anti-CSP levels or persistence. The identity of the terminal amino acid of NANP was critical, with an amino terminal Asn as in NANP or NPNA, being the best immunogens, a terminal Ala was a poor immunogen. The optimal density of the peptide per carrier was around 10 with no difference between 4 and 5 repeats. Alum adsorption enhanced antibody production to both vaccine components. Immunogenicity of an additional CSP-derived tetrapeptide, NVDP, was studied. The following peptides were synthesized: NANPNVDP2C, NVDPNANP2C and NVDP4C and bound to bromoacetylated BSA through thioether linkages at 4 to 20 chains per protein molecule. The immunogenicity of these conjugates was evaluated in young general purpose mice and IgG anti CSP measured by ELISA. For all peptides the optimal number of chains per protein molecule was between 5 and 9. The highest antibody levels were obtained using the NANPNVDP sequence but these levels did not exceed those induced by NANP only conjugates. However, combining a NANP conjugate with either NVDP or NANPNVDP conjugate increased significantly anti-CSP levels with corresponding high titers in IFA. As a next step we prepared conjugates of Pfs25-AH-Pfs25/NANP4 and Pfs25-AH-Pfs25/NVDP5 with about 4-5 peptide chains. These were injected into mice separately or as a mixture to see if the mixture induces higher anti-CSP, as was the case with BSA. A long term study to observe the antibody raise over time, is underway. We have also sequenced a plasmid carrying a modified form of the csp gene from Plasmodium vivax. This gene codes for the P. vivax CSP. The protein is characterized by a large number of repeat units made up of 8 to 11 amino acids depending on the species of Plasmodium. The reading frame of the plasmid was characterized, and the information used to produce PCR primers designed to amplify a csp gene encoding the mature P. vivax CSP, without the signal sequence and the carboxyl-terminal GPI-anchor sequence. Using these primers, we successfully amplified and cloned the modified gene into a protein expression plasmid. The plasmid was transformed into host E. coli DH5-alpha for seed stocks and E. coli BL21(DE3) for expression. The recombinant CSP was purified from inclusion bodies using 6 M urea and Ni-ion affinity chromatography. The protein was characterized by translation of the data obtained from DNA sequence analysis of the plasmid we constructed, by polyacrylamide gel electrophoresis and Western blot.